Invasive pneumococcal disease in children persists as a major cause of morbidity and mortality throughout the world despite introduction of the 7 valent capsular pneumococcal vaccine. Streptococcus pneumoniae is also the major cause of acute otitis media (AOM) which can result in hearing impairment or loss delaying the acquisition of language skills. Antimicrobial prescription for AOM is a major contributor to the rise of antimicrobial resistance. Diversifying selection-response to host immunological defenses has been a major impediment to the identification of conserved protein-based antipneumococcal vaccine candidates. A consequence of such selective pressure is the intra-species epitope variability typically found for exposed proteins on the outer surface of bacterial pathogens such as S. pneumoniae. This R21 proposal evaluates a bioinformatics-supported hypothesis for the identification of a previously unrecognized category of highly conserved, surface exposed proteins of S. pneumonia. The hypothesis is, that recombinationally quiescent 50 kb 'flanks'of ribosomal RNA operons (rrn) are a potential reservoir of genes coding for highly conserved, surface-exposed, antigenic envelope proteins (EnvP) that could be developed as potential vaccine candidates Specific aims are: (i) to utilize an innovative phylogenic strategy to screen rrn flank-encoding EnvP genes for extent of conservation across pneumococcal evolutionary diversity;(ii) clone/express/purify several such highly conserved EnvP for use as immunogens to generate polyclonal sera, and (iii) use the polyclonal antisera to test whether the EnvP meet basic criteria expected of a vaccine target: antibody accessibility of the EnvP across pneumococcal phylogenic diversity, and EnvP-elicited antibody functionality. R21-based validation of conserved, accessible and functional vaccine candidate antigens would provide the necessary preliminary results needed to support a future RO1 application focused on further EnvP vaccine development. PUBLIC HEALTH RELEVANCE: Invasive pneumococcal disease in children persists as a major cause of morbidity and mortality throughout the world despite introduction of the 7 valent capsular pneumococcal vaccine. Streptococcus pneumoniae is also the major cause of acute otitis media which can result in hearing impairment or loss, delaying the acquisition of language skills. Prevention of pneumococcal disease would have a major impact on global health and is necessary to achieve the WHO goals for reduction in childhood mortality.